Increased Microbiological Challenge Test with Bunsen Burner Inside the Cabinet

Transcription

1 Increased Microbiological Challenge Test with Bunsen Burner Inside the Cabinet Introduction Bunsen Burner is the most frequently used apparatus in the laboratory as a source of heat. Typically used inside the biological safety cabinets and laminar flow hoods for sterilizing inoculating loops, test tube lips and Petri dishes lids. This barrier is designed so that gaseous fuel may be mixed with the correct amount of air to yield the maximum amount of heat. However, placing a lighted burner into a cabinet, produces a dramatic effect. In a Class II type cabinet, the hot upflow from the burner mixed the downflowing airstreams to produce turbulence and recirculation within the working area. The notion of laminar flow may be completely destroyed and any aerosols generated beneath the burner may be carried upwards to contaminate the whole of the air within the cabinet. This is why a should not be used inside the cabinet; and an alternative technique should be found. In this experiment Esco will try to find out where the can be placed safe inside the cabinet. Experiment will be composed of three different tests: Cross contamination, Product protection and Operator protection test (KI Discus test). Each test will be done twice with different location of the inside the cabinet. Materials and Method Cabinet Model: LA2-4A2 Serial #: Motor voltage: 83.4V Nebulizer pressure: 2 psi (pressure stable) Nebulizer Serial Number: N2 Batch # of Agar: A2924 Batch # of spores: PIC22724 Vol. of Spore Suspension in Nebulizer: 55ml of 8x 6 Part I. Cross Contamination Test Procedure The LA2-4A2 with serial number biosafety cabinet was used in this experiment and was set at nominal setpoint (inflow velocity of.53m/s and downflow velocity of.35m/s). A smoke tube was used to determine the location of the inside the cabinet.

2 For the cross contamination test, the nebulizer was placed at one side of the cabinet work zone to discharge bacterial spores in the opposite direction (8x 4 of Bacillus Subtilis spores in 5 minutes). Petri dishes were placed in the work zone in a fixed arrangement. The dishes were pre-poured with sterilized Trypticase soy agar. Two rows of dishes were placed closest to the nebulizer, and this served as the control plates. Additional rows of dishes were placed with the nearest row having a distance of 4 inches from the side wall (measured from the centre of the dish). Three replicates were performed from each sides of the cabinet. Placement of the inside the cabinet Test Trial Flame Height (cm) Distance from the Side wall (cm) Distance from the Back wall (cm) Location # Location # Acceptance Criteria The total number of CFU recovered on all agar plates from 4 inches and beyond shall not exceed 2 Colony Forming Units per test. The control plate will be considered positive when it contains more than 3 CFU of bacteria. Calculation for concentration of spore suspension To obtain the required spore solution for nebulizer (product protection test): ml of the original suspension was transferred to the first dilution tube containing 9 ml of sterile deionized water (tube, 8 ). Another ml from the first tube was transferred to the second dilution tube with 9 ml sterile deionized water (tube 2, 6 ). From tube 2, 6 ml of the diluted suspension was obtained and added to the third dilution tube containing 54 ml of sterile deionized water. To get the target concentration of 8x 6. 79x 6 x A = 8x 6 (target) => 79A = 8 => 79A = 48 => A = 6mL A + (6 A) 6 Therefore mix Spore suspension (A) : 6mL and sterile deionized water: 54 ml In this particular test, 79x 9 spores per ml were obtained.

3 Experiment Set-up Location #

4 Location #2

5 Results Location # Nebulizer on Left Side of Work Space Test Recovery of CFU Control 4 mark and beyond Plates 4 plates CFU 3 plates TNTC 6 plates 6 to 3 CFU Total CFU 2 4 plates CFU 3 plates TNTC 6 plates 5 to 3 CFU Total CFU 3 4 plates CFU 3 plates TNTC 6 plates 3 to 3 CFU Total CFU Nebulizer on Right Side of Work Space Test Recovery of CFU Control 4 mark and beyond Plates 4 plates CFU plate TNTC 6 plates to 2 CFU Total CFU 2 4 plates CFU plate TNTC 6 plates to 2 CFU Total CFU 3 4 plates CFU plate TNTC 6 plates to 2 CFU Total CFU Location #2 Nebulizer on Right Side of Work Space Test Recovery of CFU Control 4 mark and beyond Plates 7 plates have >CFU 9 plates TNTC 5 plates have to 8 CFU Total >3 CFU (Fail) 2 7 plates have >CFU 9 plates TNTC 5 plates have to 2 CFU Total >3 CFU (Fail) 3 7 plates have >CFU 9 plates TNTC 5 plates have to 23 CFU Total >3 CFU (Fail)

6 Illustration of Results Location # Test I. Left Side of Work Space Right Side of Work Space Test II. Left Side of Work Space Right Side of Work Space Test III. Left Side of Work Space Right Side of Work Space

7 Location #2 Test I. Right Side of Work Space Test II. Right Side of Work Space > > 2 > > > 7 > > > > > > > > > 4 inch line 4 inch line Test III. Right Side of Work Zone > > > > > > > 4 inch line

8 Part II. Product Protection Test Procedure The LA2-4A2 with serial number biosafety cabinet was used in this experiment and was set at nominal setpoint (inflow velocity of.53m/s and downflow velocity of.35m/s). A smoke tube was used to determine the location of the inside the cabinet. For the product protection test, the cabinet was set-up with Petri dishes in the work zone. The dishes were pre-poured with sterilized Trypticase soy agar. A fixed amount of bacterial spores was discharged from the nebulizer (8x 6 of Bacillus Subtilis spores for 5 minutes). The stainless steel cylinder (acts as an artificial arm to simulate normal operating conditions, airflow disturbance) was placed at the centre of the working area. A single Petri dish was placed beneath the front air grille (supported by an empty Petri dish) and serve as a control. Placement of inside the cabinet Test Trial Flame Height (cm) Distance from the Side wall (cm) Distance from the Back wall (cm) Location # Location # Acceptance Criteria The maximum number of Colony Forming Units (CFU) in total recovered from all agar plates in the work zone shall not exceed 5. The control plate will be considered positive when it contains more than 3 CFU of bacteria. Calculation for concentration of spore suspension To obtain the required spore solution for nebulizer (product protection test): ml of the original suspension was transferred to the first dilution tube containing 9 ml of sterile deionized water (tube, 8 ). Another ml from the first tube was transferred to the second dilution tube with 9 ml sterile deionized water (tube 2, 6 ). From tube 2, 6 ml of the diluted suspension was obtained and added to the third dilution tube containing 54 ml of sterile deionized water. To get the target concentration of 8x 6. 79x 6 x A = 8x 6 (target) => 79A = 8 => 79A = 48 => A = 6mL A + (6 A) 6 Therefore mix Spore suspension (A) : 6mL and sterile deionized water: 54 ml In this particular test, 79x 9 spores per ml were obtained.

9 Results Location # Test No. of CFU from work area Positive Control Plates All plates has CFU TNTC Total CFU (Pass) 2 All plates has CFU TNTC Total CFU 3 All plates has CFU TNTC Total CFU Location #2 Test No. of CFU from work area plate has 5 CFU TNTC 2 plates has 4 CFU plate has 3 CFU 7 plates has 2 CFU 8 plates has CFU Total 48 CFU (Failed) Positive Control Plates Illustration of Results Location # Test I Nebulizer

10 Test II Nebulizer Test III Nebulizer Location #2 Nebulizer

11 Part III. Operator Protection Test (KI Discus Test) Procedure The LA2-4A2 with serial number biosafety cabinet was used in this experiment and set at nominal setpoint (inflow velocity of.53m/s and downflow velocity of.35m/s). A smoke tube was used to determine the location of the inside the cabinet. For the personnel/operator protection test, KI discus was used, according to study, this is more stringent than the microbiological test method, moreover, more difficult to pass. The KI Discus was positioned in front of the cabinet and the stainless steel cylinder was placed in the centre of the work tray to simulate normal operating conditions (the airflow disturbance of operator's arm). The disc was placed inside the work zone to discharge the Potassium iodide spray outwards. Four (4) suction cones were placed outside the cabinet as it simulate the breathing of the operator working on the cabinet. The suction cone has a filter membrane inside to suck the Potassium iodide released by the disc. Placement of inside the cabinet Test Trial Flame Height (cm) Distance from the Side wall (cm) Distance from the Back wall (cm) Location # Location # Acceptance Criteria The total number of brown dots recovered from each filter membrane shall not exceed more than 62. This will correspond to aperture protection factor of, meaning that for every, particles liberated behind the sash window, only manages to escape. If there are zero brown dots captures, this translates to aperture protection factor of greater than 6,2,. Results Location # X Y X Y Location #2 X 2 Y 9 X 4 Y 23

12 Conclusion, alcohol lamp, or any other apparatus which requires the consumption of flames, should not be used inside biosafety cabinets. Flame creates turbulence in airflow and will compromise sterility and heat build-up may damage the filters. When seemed absolutely necessary, should be place at the rear of the workplace where resulting air turbulence has a minimal effect. In this particular experiment, result showed that the safest location of the or any fire-requiring material inside the cabinet would be at the further right side of the cabinet: 7. cm from the right side wall and 2.5 cm from the back wall. Microbiological test result showed no bacterial growth on the agar plates. Hence, this location in the cabinet passed the three (3) microbiological tests which are the Cross contamination, Product protection and Operator protection test.